Detection of Binding
Detecting and quantifying interactions such as binding between biomolecules is of central interest in modern molecular biology and medicine. Ligand and drug binding to molecules is an important theme in modern biology, medicine and drug development. Binding reactions between biological components often result in a conformational or dipole moment (or induced dipole moment) change. Conformational change (a change in orientation or position of a molecule or subpart(s) of a molecule) is an essential feature of signaling in many systems. A technique for detecting activation of a signal transducer (e.g., a receptor in a membrane), through a direct measure of conformational change or change in dipole moment, would be of value in basic research and drug discovery. For example, high-throughput drug screening for potential agonists or antagonists of a receptor can be carried out using the present invention as a method for detection of whether potential agonists or antagonists induce a conformational change—indicative of binding and activation of a receptor. Because the conformational change is a direct indicator of receptor activation, it is an excellent means of screening for drugs; current techniques often rely on indirect measures of activation such as changes in fluorescence intensity that are concomitant with changes in Ca2+ ion concentration, which in turn is caused by the receptor activation. Current techniques that directly measure activation, such as patch-clamping techniques with an ion channel protein, are not amenable to high-throughput scaleup and require a skilled technician to operate, resulting in higher cost to the user.
Fluorescence-based or surface plasmon-based detection are used to detect binding interactions, with varying success and efficiency. Problems using fluorescence include the presence of a natural fluorescent background in many (non-labeled) biological samples as well as photobleaching. Detection of orientational changes accompanying target binding is difficult to do using fluorescence as the technique is not very sensitive to label orientation: fluorescence polarization, not being a coherent technique, is sensitive to rotational motion during the fluorescence lifetime that makes it difficult to assign small measured changes in a particular polarization direction to conformational changes (rather than due to rotational motion). It is also difficult to separate small changes in probe orientation from the large fluorescent background that may be present in many biological samples. Furthermore, it is often not trivial to separate a signal indicative of binding from one indicative of a receptor activation using fluorescence; for example, fluorescently tagged targets are tested for their ability to bind to a given receptor using fluorescence polarization—but binding of a target to a surface such as a cell surface can occur non-specifically such that the target does not specifically bind to the receptor of interest, yet can still give a signal indicative of said binding; direct detection of conformational change as an indicator of receptor activation is a more direct means of assaying for activation. Wavelength-based changes due to changes in the microenvironment of the label as a result of conformational change can be followed, but not all conformational changes lead to a change in microenvironment and it may be difficult to assign relative changes due to microenvironment to the degree of conformational change that actually occurs. Examples of fluorescent-based prior art to detect conformational changes in proteins include the following: Mannuzzu et al. (1996) Science 271, 213-216 describe the direct physical measure of conformational rearrangement underlying potassium channel gating using an fluorescently tagged channel protein. Gether et al., “Fluorescent labeling of purified P2 adrenergic receptor,” J. Biol. Chem. 270, 28628-28275 (1995), describe the fluorescent labeling of soluble purified 02 adrenergic receptor to detect ligand-specific conformational changes. Turcatti et al., “Probing the Structure and Function of the Tachykinin Neurokinin-Receptor through Biosynthetic incorporation of fluorescent amino acids at specific sites,” J. Biol. Chem. 271, 19991-19998, (1996), describe the incorporation of non-natural fluorescent amino acids into a receptor to monitor ligand binding. Liu et al., “Site-Directed fluorescent labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains,” Biochemistry 35, 11865-11873, (1996), describe fluorescent labeling of soluble purified P-glycoprotein to detect ligand binding.
Fluorescence has also been used to detect binding of targets to ion channel receptors when the binding leads to a change in transmembrane potential in cells. The transmembrane potential change (e.g., a depolarization) leads to a change in the intensity, lifetime, wavelength, etc. of the fluorescent or nonlinear-active label in the membrane. Apart from various problems in the detection itself—concerning photobleaching, artifacts and background noise, these methods only provide indirect assays for the probe-target binding. For example, it is possible that a given target binds to ion channel probes and the ion channels are activated, but that this does not lead to a change in transmembrane potential, either for natural reasons or because there are problems in the cells in the normal mechanism for producing the change in transmembrane potential. It is desirable therefore to have a direct, optical means of detecting probe-target binding reactions in cases where the binding reaction results in a change in orientation or conformation of the probe.
Molecular Beacons
Molecular beacons are also used for the detection of binding interactions. A molecular beacon (MB) probe is well known in the art as a hairpin-loop, single-stranded oligonucleotide comprising a probe sequence embedded within complementary sequences that form a hairpin stem. The loop portion of the molecule can form a double-stranded DNA in the presence of complementary nucleic acid. A fluorophore is covalently attached to one end of the oligonucleotide, and a nonfluorescent quencher is covalently attached to the other end. There are typically five to eight bases at each side of the two ends of the beacon which are complementary to each other. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. When the beacon binds to its target, the rigidity of the probe-target duplex forces the stem to unwind, which causes the separation of the fluorophore and the quencher and the restoration of fluorescence. This permits the detection of probe-target hybrids in the presence of unhybridized probes.
The following (and references therein) describe the production, design and use of molecular beacons (MBs) in the fluorescence literature:    Sequence Dependent Rigidity of Single Stranded DNA, Phys. Rev. Lett., 85, 2400 No. 11, Noel L. Goddard, 1 Gregoire Bonnet, 1 Oleg Krichevsky, 2 and Albert Libchaber, 2000.    Metal-containing DNA hairpins as hybridization probes, H. S. Joshi and Y. Tor, Chem. Commun., 2001, 549-550.    Spectral Genotyping of Human Alleles, L. G. Kostrikis, S. Tyagi, M. M. Mhlanga, D. D. Ho, F. R. Kramer, Science v. 279, February 1998.    Molecular Beacons for DNA Biosensors with Micrometer to Submicrometer Dimensions, X. Liu, W. Farmerie, S. Schuster, W. Tan, Anal. Biochem., 283, 56-63, 2000.    Multiplex detection of single-nucleotide variations using molecular beacons, S. A. E. Marras, F. R. Kramer, S. Tyagi, Genetic Analysis: Biomolecular Engineering, 14 (1999), 151-156.    Screening unlabeled DNA targets with randomly ordered fiber-optic gene arrays, F. J. Steemers, J. A. Ferguson, D. R. Walt, Nature Biotechnology, v. 18, 2000, 91.    Wavelength-shifting molecular beacons, S. Tyagi, S. A. E. Marras, F. R. Kramer, Nature Biotechnology, v. 18, 2000, 1191.    Molecular Beacons: Probes that Fluoresce upon Hybridization, S. Tyagi, F. R. Kramer, Nature Biotechnology, v. 14, 1996, 303.    Technology for microarray analysis of gene expression, A. Watson, A. Mazumder, M. Stewar, S. Balasubramanian, Current Opinion in Biotechnology, 9, 609-614, 1998.    Design of a Molecular Beacon DNA Probe with Two Fluorophores, P. Zhang, T. Beck, W. Tan, Angew. Chem. Int. Ed. 40, 402, 2001    Bonnet G, Tyagi S, Libchaber A, and Kramer F R (1999) Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc Natl Acad Sci USA 96, 6171-6176.    Dubertret B, Calame M, and Libchaber A (2001) Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat Biotechnol 19, 365-370.    Marras S A E, Kramer F R, and Tyagi S (1999) Multiplex detection of single-nucleotide variations using molecular beacons. Genet Anal 14, 151-156.    Mullah B and Livak K (1999) Efficient automated synthesis of molecular beacons. Nucleosides & Nucleotides 18, 1311-1312.    Ortiz E, Estrada G, and Lizardi P M (1998) PNA molecular beacons for rapid detection of PCR amplicons. Mol Cell Probes 12, 219-226.    Tyagi S, Marras S A E, and Kramer F R (2000) Wavelength-shifting molecular beacons. Nat Biotechnol 18, 1191-1196.    Tyagi S, Bratu D P, and Kramer F R (1998) Multicolor molecular beacons for allele discrimination. Nat Biotechnol 16, 49-53.    Tyagi S and Kramer F R (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14, 303-308.    El-Hajj H H, Marras S A, Tyagi S, Kramer F R, and Alland D (2001) Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons. J Clin Microbiol 39, 4131-4137.    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Van Schie R C, Marras S A, Conroy J M, Nowak N J, Catanese J J, and de Jong P J (2000) Semiautomated clone verification by real-time PCR using molecular beacons. Biotechniques 29, 1296-1306.    Vet J A, Majithia A R, Marras S A E, Tyagi S, Dube S, Poiesz B J, and Kramer F R (1999) Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc Natl Acad Sci USA 96, 6394-6399.    Xiao G, Chicas A, Olivier M, Taya Y, Tyagi S, Kramer F R, and Bargonetti J (2000) A DNA damage signal is required for p53 to activate gadd45. Cancer Res 60, 1711-1719.    Zhang L, Lewin S R, Markowitz M, Lin H H, Skulsky E, Karanicolas R, He Y, Jin X, Tuttleton S, Vesanen M, Spiegel H, Kost R, van Lunzen J, Stellbrink H J, Wolinsky S, Borkowsky W, Palumbo P, Kostrikis L G, and Ho D D (1999) Measuring recent thymic emigrants in blood of normal and HIV-1-infected individuals before and after effective therapy. J Exp Med 190, 725-732.    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Zhang P, Beck T, and Tan W (2001) Design of a molecular beacon DNA probe with two fluorophores. Angew. Chem. Int. Ed. 40, 402-405.Nonlinear Optical Techniques
Surface-selective nonlinear optical techniques have previously been confined mainly to physics and chemistry since relatively few biological samples are intrinsically non-linearly active. Examples include the use of an optically nonlinear-active dye as a membrane stain—and an endogenous nonlinear-active stain (GFP) that is used to image biological cells (Campagnola et al., “High-resolution nonlinear optical imaging of live cells by second harmonic generation,” Biophysical Journal 77 (6), 3341-3349 (1999), Peleg et al., “Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites,” Proc. Natl. Acad. Sci. V. 96, (1999), 6700-6704 and references therein). The following references (and references therein) are exemplary of this art:    A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouevitch, Z. Rothman, L. Loew and M. Sheves, “Second Harmonic Generation of Biological Interfaces: Probing the Membrane Protein Bacteriorhodopsin and Imaging Membrane Potential Around GFP Molecules at Specific Sites in Neuronal Cells of C. elegans,” Chemical Physics 245, 133 (1999).    O. Bouevich, A. Lewis, I. Pinnevsky and L. Loew, “Probing Membrane Potential with Non-linear Optics,” Biophys. J. 65, 672 (1993).    I. Ben-Oren, G. Peleg, A. Lewis, B. Minke and L. Loew, “Infrared nonlinear optical measurements of membrane potential in photoreceptor cells,” Biophys. J. 71, 616 (1996).    G. Peleg, A. Lewis, M. Linial and L. M. Loew, “Non-linear Optical Measurement of Membrane Potential Around Single Molecules at Selected Cellular Sites,” Proc. Acad. Sci. USA 96, 6700 (1999).    P. Campagnola, Mei-de Wei, A. Lewis and L. Loew, “High-Resolution Nonlinear Optical Imaging of Live Cells by Second Harmonic Generation,” Biophys. J. 77, 3341 (1999).    J. Y. Huang, A. Lewis and L. Loew, “N[on-linear Optical Properties of Potential Sensitive Styryl Dyes”, Biophysical J. 53, 665 (1988).    A. Lewis, A. Khatchatouriants, M. Treinin, Z. Chen, G. Peleg, N. Friedman, O. Bouevitch, Z. Rothman, L. Loew and M. Sheves, “Second Harmonic Generation of Biological Interfaces: Probing Membrane Proteins and Imaging Membrane Potential Around GFP Molecules at Specific Sites in Neuronal Cells of C. elegans,” Chemical Physics 245, 133-144 (1999).    A. Khatchatouriants, A. Lewis, Z. Rothman, L. Loew and M. Treinin, “GFP is a Selective Non-Linear Optical Sensor of Electrophysiological Processes in C. elegans,” Biophys. J. (in press, 2000)
In the prior art, nonlinear-active stains are immobilized in membranes and these stains are used to image the cell surfaces. However, the stains intercalate into the membranes in either an ‘up’ or ‘down’ direction, thus reducing the total nonlinear signal due to destructive interference. Nonlinear optically active dyes have also been used to measure the kinetics of those dyes crossing lipid bilayers in liposomes (A. Srivastava and K B Eisenthal, “Kinetics of molecular transport across a liposome bilayer,” Chem. Phys. Lett. 292 (3): 345-351 (1998)).
The present invention provides advantages over fluorescence-based detection, such as reduced background, reduced photobleaching, simplified optical detection, and no need for labor- or time-consuming washing steps. These advantages are due to the low background of the nonlinear optical techniques and the fact that the method is a scattering rather than an absorption process.
It is therefore an object of the present invention to provide a direct, optical means of detecting probe-target binding reactions in cases where the binding reaction results in a change in orientation or conformation of the probe, target, or both probe and target.